The papillomaviruses cause benign tumors of epithelial cells in both humans and animals, and is some cases these tumors progress to malignancies. The viruses are poorly understood because they do not productively infect cells in tissue culture, but mouse cells in culture can be oncologically transformed by bovine papillomavirus type I (BPV) or by BPV DNA. We will continue our comprehensive genetic analysis of bovine papillomavirus. Our long range goals are to identify the viral genes and gene product and to characterize their biological activities, to established the mechanism of their regulation and to determine their biochemical contribution to oncogenic transformation. The primary approach that will be used is the construction and analysis of viral mutants. A variety of directed mutagenesis techniques will be used to introduce defined mutations into each of the viral genes and regulatory signals. Initially, we will concentrate on three BPV gene ORFS E5, E2, and E6 which seem to influence the ability of the viral DNA to transform mouse cells or the phenotype of the transformed cells. The effect of the mutations will be determined in a variety of biological and biochemical assays in mouse C127 cells and in other cultured epithelial cells. Cell lines that express a subset of the viral genes will be generated and analyzed to identify viral gene products and their functions. Transient assays systems will be developed to investigate papillomavirus gene regulation. Temperature sensitive mutants will also be isolated and analyzed. We will also attempt to induce capsid protein synthesis in cultured cells so that viral mutants can be encapsidated. This ability would enable us to complement the analysis of the in vitro activities of the mutants with studies of their activities during lytic growth in animals. These studies should help indicate how each viral gene contributes to the conversion of a normal cell into a malignant one. Because of the association of papillomavirus with human carcinomas, our findings may be relevant to understanding the genesis of human cancers.